R RECEPTORS AND CELL SIGNALING, unit from cholesterol involves the hydroxylation of C-20 INTRACELLULAR RECEPTORS —STEROID and C-22 followed by cleavage by desmolase to produce HORMONES AND NO pregnenolone (C21). Conversion of pregnenolone to pro- gesterone requires oxidation of the 3-hydroxyl group to SCOTT L. DIAMOND a 3-keto group and isomerization of the C-5-C-6 dou- Institute for Medicine and Engineering ble bond to the C-4-C-5 position. Progesterone can be Philadelphia, Pennsylvania metabolized to the major glucocorticoid, cortisol (by mul- tiple hydroxylations), or to the major mineralocorticoid, aldosterone (by multiple hydroxylations and oxidation of OUTLINE the C-18 methyl group to an aldehyde). Alternatively, progesterone can be modified to androgens and estrogens Introduction via specific hydroxylation, chain cleavage, and reduction Steroids and their Biosynthesis reactions. Thyroid hormones, retinoids, and vitamin D are Steroid Receptors and Gene Regulation not derived from cholesterol and are unrelated in struc- ture to steroid hormones, but also diffuse across the cell Nitric Oxide and Nitric Oxide Synthase membrane to bind their intracellular receptor in target Nitric Oxide: cGMP-Dependent Signaling tissues. Nitric Oxide: cGMP-Independent Signaling Nitric Oxide, Peroxynitrite, and Nitrotyrosine STEROID RECEPTORS AND GENE REGULATION Crosstalk between Nitric Oxide and Steroid Signaling Summary: Relevance to Biotechnology Steroid regulation of gene expression involves steroid Acknowledgments molecules crossing the plasmalemma via passive transport Bibliography (Fig. 1). Once in the cytoplasm, the steroid binds its receptor with high affinity (Kd ~ 1 nM), which causes the release of chaperones [including heat shock proteins hsp70 INTRODUCTION and hsp90, p23, and various peptidylprolyl isomerase (PPIase)-active immunophilins/cyclophilins (CyP) such as Soluble extracellular molecules that cross the cell FKBP52 (hsp56), CyP18, and CyP40] from the receptor, membrane, bind an intracellular receptor, and alter and consequent exposure of nuclear localization sequences cellular function are essential regulators of mammalian and phosphorylation sites in the steroid receptor (1,2). The endocrine biology. This article reviews two of the most chaperones maintain the receptor in a conformation that well-studied and prototypical intracellular receptors: is competent for steroid binding but lacks DNA regulatory steroid hormone receptors, which are transcriptional activity. The receptor with bound steroid (termed the regulators, and the nitric oxide receptor, which is steroid receptor complex or liganded steroid receptor) soluble guanylate cyclase. These two systems are quite undergoes nuclear import, after which the liganded steroid different and represent the diverse mechanisms by which receptor dimerizes. The dimer binds its specific hormone animal cells respond to diffusible mediators that cross response element (HRE or SRE for steroid response the plasmalemma. While steroids alter gene and cell element) to activate or repress within 30 min about 50 regulation on a time scale of minutes to days, nitric to 100 primary response genes via its interactions with oxide (NO) has diverse effects (on a time scale of numerous other transcriptional factors and consequent seconds to hours) such as vasodilation, inhibition of changes in chromatin structure. Other secondary response platelet activation, neurotransmission, and cytotoxicity. In genes can then be regulated over the course of hours or days some instances, crosstalk exists between NO and steroid through mechanisms requiring expression of the primary receptor pathways. The biochemistry and molecular response genes. The ability of a liganded steroid receptor to biology of steroids and NO can impact numerous find the handful of promoters containing HRE among the biotechnological applications such as gene therapy, tissue ~ 100,000 genes in the mammalian genome represents an engineering, and bioreactor dynamics, particularly in extraordinary diffusional search process. The dimer may relation to the external control of cellular metabolism undergo one-dimensional diffusion along DNA segments and gene expression. or DNA-intersegment transfer facilitated by the bivalent interactions made possible with a dimer complex (3). The steroid receptor has three functional domains: the STEROIDS AND THEIR BIOSYNTHESIS N-terminal domain (site of transcriptional activation and binding of transcriptional factors; A/B domain in Table 1), Steroid hormones are small hydrophobic metabolites a DNA binding domain (which also confers dimerization; of cholesterol (C27), and are broadly divided into five C/D domain in Table 1), and a hormone binding domain classes (Table 1): progestagens, glucocorticoids, mineralo- at the C-terminus of the protein which also participates corticoids, androgens, and estrogens. Cleavage of the C6 in transcriptional activation (E domain in Table 1) (4). 1 17 Table 1. Steroids and their receptors Steroid Receptor Response element Steroid structure Progestagens (C21) Dimerization Same as GRE Pregnenolone 17-hydroxypregnenolone Progesterone PR 17-hydroxyprogesterone DNA Ligand transactivation Progesterone Glucocorticoids (C21) GR GRE Corticosterone 5'AGAACAnnnTGTTCT-3 Deoxycorticosterone Cortisol 11-Deoxycortisol Cortisone 18-hydroxy corticosterone 1-a-hydroxycorticosterone Aldosterone Cortisol Mineralocorticoids (C21) MR Same as GRE Aldosterone Aldosterone Androgens (C 19) AR Same as GRE 1 l-/*-hydroxyandrostene dione 5-a-Androstanediol Androsterone Epiandrosterone Andronosterone Testosterone Testosterone Epitestosterone 5-a-dihydrotestosterone 5-/?-dihydrotestosterone 11-keto-testosterone Estrogens (Cis) ER ERE Estrogen 5'-PuGGTCAmInTGACCPy-^ Estrone Estradiol Estriol Estradiol The DNA binding domain and hormone binding domain three-dimensional assemblies with general transcription are highly conserved across the steroid receptor family, factors (TBP, TAF//30, dTAF//110, TFIIB), coactivators while the N-terminus, which controls transcriptional (AEA70, RIP140, ERAP, TIF-2, SRC-I, RSP5, TIFl, regulatory activity, is not. Crystal structures are widely SUGl, GRIP170), sequence-specific transcriptional factors available for many mammalian steroid receptors (see (fos/jun, GATA-I, ReIA, PML, OTF-I), and chromatin http: 11nrr.georgetown.edu /NRR /NRR.html). regulators (hBrm, BRGl, Spt6, and HMGl) (5-11). The liganded steroid receptor dimer bound to its HRE Coactivators can bridge the dimer with other DNA binding has an extremely complicated and not fully characterized proteins of the transcriptional initation complex that set of interactions with other proteins. Once in the assemble on the TATA box of the promoter (Fig. 1). nucleus, steroid hormone receptors can interact in In contrast to activation, gene repression is commonly steroid hormone hsp90,hsp70 hsp56, others steroid CYTOPLASM receptor with caperones NUCLEUS sequence-specific coactivators transcriptional activators transcription chromatin HRE TATA structure transcription initiation factors, RNA polymerase Figure 1. Steroid receptor function. viewed as an action of the steroid hormone dimer that: sequence are possible. A promoter may contain several (i) sterically blocks DNA binding sites used by other HREs. Receptors for glucocorticoids, mineralocorticoids, factors, such as fos/jun (API complex); (2) inactivates progesterone, and androgens utilize the same 6-base DNA normally competent transcription factors; or (3) serves as sequence 5'-AGAACA-S' (4). The estrogen receptor utilizes a binding sink for factors needed for transcription (4). the sequence 5'-AGGTCA-S', which is also the sequence In addition to these issues of assembly on the promoter, used by nonsteroid nuclear receptors. The DNA binding steroid receptors may function by altering the structure domain of the steroid receptor contains two zinc binding of the chromatin via its effects on hBrm and BRGl motifs —the P Box binds the HRE half-site and the D (5-7). hBrm is the human homolog of Drosophila Box facilitates dimerization. Liganded steroid receptors Brama (brm) and yeast SWI/SNF complex and is a predominantly form homodimers on the HRE in vivo. 180-kDa nuclear protein that mediates ATP-dependent The nonsteroidal thyroid hormone response elements nucleosome disruption. BRGl (205 kDa) is a second contain copies of a core consensus motif: 5'-AGGTCA- human homolog of yeast SWI/SNF that binds the 3' commonly in a head-to-tail repeat separated by four retinoblastoma gene product Rb, on which assembly base or inverted tail-to-tail repeats. Numerous variations processes occur. Interestingly, hBrm is not expressed in exist naturally or have been constructed artificially. The all cells. response elements for one or more hormone receptors may The human progesterone receptor (PR) can bind steroid exist on a single promoter to cause synergistic activation receptor coactivator 1 (SRC-IA) for up-regulation of tran- of the gene. In promoter construct experiments, a scription. Interestingly, SRC-IA has histone acetyltrans- single glucocorticoid response element (GRE) (or estrogen ferase activity. Histone acetylation opens up the chromatin response element, ERE) upstream of a TATA box confers a structure for transactivating factors. Human PR can also steroid responsive promoter, with two GRE (or two ERE) bind p300/CREB-binding protein (CBP)-associated factor, elements resulting in synergistic gene activation. Such which has histone acetyltransferase activity. The localiza- experiments